Hepatitis C virus modified sE2F442NYT as an antigen in candidate vaccine facilitates human immune cell activation.

The rational selection of hepatitis C virus (HCV) vaccine antigen will aid in the prevention of future chronic liver disease burden and associated healthcare costs. We have previously shown that HCV E2 glycoprotein is not highly immunogenic, and the modification of E2 reduced CD81 binding and displayed altered cytokine and protective immune responses in vitro and in a surrogate mouse model. Here, we compared the influence of a parental and a modified sE2F442NYT glycoprotein region from HCV genotype 1a for the activation of peripheral blood mononuclear cell (PBMC)-derived dendritic cells (DCs), CD4+T cells, and B cells. Modified sE2F442NYT, when incubated with DCs, induced a higher number of CD86-positive cells. The sE2F442NYT or parental sE2 encapsulated as mRNA-lipid nanoparticle (sE2F442NYT mRNA-LNP) primed DCs co-cultured with autologous CD4+T cells did not induce CD25 or forkhead box P3 expression. PBMC-derived CD4+T cells treated with sE2F442NYT exhibited enhanced signal transducer and activator of transcription (Stat)1/Stat4 phosphorylation in response to anti-CD3/CD28 stimulation in comparison to parental sE2 treatment and facilitated isotype switching in B cells, leading to the generation of a broader subclass of antibodies. Cells treated with modified sE2F442NYT displayed an increase in activated Stat3 and extracellular signal-regulated kinase (ERK). Likewise, PBMC-derived naïve B cells upon in vitro stimulation with sE2F442NYT induced an increased proliferation, Stat3 and ERK activation, and protein kinase B (Akt) suppression. Thus, the modified sE2F442NYT antigen from HCV facilitates improved DC, CD4+T, and B cell activation compared to parental sE2 to better induce a robust protective immune response, supporting its selection as an HCV candidate vaccine antigen for preclinical and clinical HCV vaccine trials.IMPORTANCEThe nature of an enhanced immune response induced by sE2F442NYT will help in the selection of a broad cross-protective antigen from hepatitis C virus genotypes, and the inclusion of relatively conserved sE1 with sE2F442NYT may further strengthen the efficacy of the candidate vaccine in evaluating it for human use.

mRNA-LNP vaccine-induced CD8+ T cells protect mice from lethal SARS-CoV-2 infection in the absence of specific antibodies.

The role of CD8+ T cells in SARS-CoV-2 pathogenesis or mRNA-LNP vaccine-induced protection from lethal COVID-19 is unclear. Using mouse-adapted SARS-CoV-2 virus (MA30) in C57BL/6 mice, we show that CD8+ T cells are unnecessary for the intrinsic resistance of female or the susceptibility of male mice to lethal SARS-CoV-2 infection. Also, mice immunized with a di-proline prefusion-stabilized full-length SARS-CoV-2 Spike (S-2P) mRNA-LNP vaccine, which induces Spike-specific antibodies and CD8+ T cells specific for the Spike-derived VNFNFNGL peptide, are protected from SARS-CoV-2 infection-induced lethality and weight loss, while mice vaccinated with mRNA-LNPs encoding only VNFNFNGL are protected from lethality but not weight loss. CD8+ T cell depletion ablates protection in VNFNFNGL but not in S-2P mRNA-LNP-vaccinated mice. Therefore, mRNA-LNP vaccine-induced CD8+ T cells are dispensable when protective antibodies are present but essential for survival in their absence. Hence, vaccine-induced CD8+ T cells may be critical to protect against SARS-CoV-2 variants that mutate epitopes targeted by protective antibodies.

Metal Hydride Vibrations: The Trans Effect of the Hydride.

trans-Dihydride complexes are important in many homogeneous catalytic processes. Here vibrational spectroscopy and density functional theory (DFT) methods are used for the first time to reveal that 4d and 5d metals transmit more effectively than the 3d metals influence of the ligand trans to the hydride and also couple the motions of the trans-hydrides more effectively. This property of the metal is linked to higher hydride reactivity. The IR and Raman spectra of trans-FeH2(dppm)2, trans-RuH2(PPh(OEt)2)4, and mer-IrH3(PiPr2CH2pyCH2PiPr2) provide M-H force constants and H-M-H interaction force constants that increase as FeII < RuII < IrIII. DFT methods are used to determine, for the first time, the effect of the metal ion (MnI, ReI, FeII, RuII, OsII, CoIII, RhIII, IrIII, PtIV) and ligands on the gap in wavenumbers between the symmetric νsymH-M-H and antisymmetric νasymH-M-H vibrational modes of hydrides that are mutually trans in d6 octahedral complexes. The magnitude of this gap reflects the degree of coupling of, or interaction between, these modes, and this is shown to be a distinctive property of the metal ion. The more polarizable 4d and 5d metal ions are found to have an average gap of 246 cm-1, while the 3d metals have only 90 cm-1. This has been verified experimentally for 3d, 4d, and 5d transition-metal trans-dihydrides, where both the IR and Raman spectra have been measured: trans-RuH2(PPh(OEt)2)4 (from the literature) and trans-FeH2(PPh2CH2PPh2)2 and mer-IrH3(PiPr2CH2pyCH2PiPr2) (this work). Because the 4d and 5d metal ions tend to be better catalysts for the hydrogenation of substrates with polar bonds, this gap may be a fundamental determinant of the kinetic hydricity of the catalyst. Finding the magnitude of this gap and a new estimate of the large hydride trans-effect (Δνt -235 cm-1) allows us to improve the simple equation reported previously, which allows a better estimate of νM-H.

Density Functional Theory Calculations Support the Additive Nature of Ligand Contributions to the pKa of Iron Hydride Phosphine Carbonyl Complexes.

The acid dissociation constant Ka of a transition-metal hydride complex is a key thermodynamic quantity for evaluating reactivity and stability of the complexes and their conjugate bases in stoichiometric and catalytic reactions. It can be estimated using a simple ligand acidity constant (LAC) empirical equation for a wide range of complexes. Here, we provide the first density functional theory (DFT) study that supports the additive nature of ligand contributions to the pKa of metal hydride complexes. Specifically, the pKa values of iron hydride complexes [FeH(CO)x(PR3)(5-x)]+ in either tetrahydrofuran or dichloromethane solutions are estimated using the LAC method and DFT calculations. There is a linear correlation between these two methods, and both predict a surprisingly linear increase in pKa over a wide range from approximately -15 for x = 5 to approximately 40 for x = 0. The LAC equation predicts that pKaTHF or pKaDCM increases by 9 units with the replacement of each CO ligand with a trialkylphosphine ligand in a stepwise fashion, whereas the DFT calculations predict the step size will be approximately 11. The two methods agree with pKDCM data available for x = 3 and qualitative data for x = 1 and 0, but further quantitative measurements over a wider range are needed to firmly establish the trend. The free energy of protonation and the energy of the highest occupied molecular orbital of Fe(CO)x(PR3)(5-x) (mainly nonbonding d electrons) increase linearly with phosphine substitution, and this increases the pKa value as observed.

Antibodies to Crucial Epitopes on HSV-2 Glycoprotein D as a Guide to Dosing an mRNA Genital Herpes Vaccine.

The toxicity of mRNA-lipid nanoparticle (LNP) vaccines depends on the total mRNA-LNP dose. We established that the maximum tolerated dose of our trivalent mRNA-LNP genital herpes vaccine was 10 µg/immunization in mice. We then evaluated one of the mRNAs, gD2 mRNA-LNP, to determine how much of the 10 µg total dose to assign to this immunogen. We immunized mice with 0.3, 1.0, 3.0, or 10 µg of gD2 mRNA-LNP and measured serum IgG ELISA, neutralizing antibodies, and antibodies to six crucial gD2 epitopes involved in virus entry and spread. Antibodies to crucial gD2 epitopes peaked at 1 µg, while ELISA and neutralizing titers continued to increase at higher doses. The epitope results suggested no immunologic benefit above 1 µg of gD2 mRNA-LNP, while ELISA and neutralizing titers indicated higher doses may be useful. We challenged the gD2 mRNA-immunized mice intravaginally with HSV-2. The 1-µg dose provided total protection, confirming the epitope studies, and supported assigning less than one-third of the trivalent vaccine maximum dose of 10 µg to gD2 mRNA-LNP. Epitope mapping as performed in mice can also be accomplished in phase 1 human trials to help select the optimum dose of each immunogen in a multivalent vaccine.

Development of a pentavalent broadly protective nucleoside-modified mRNA vaccine against influenza B viruses.

Messenger RNA (mRNA) vaccines represent a new, effective vaccine platform with high capacity for rapid development. Generation of a universal influenza virus vaccine with the potential to elicit long-lasting, broadly cross-reactive immune responses is a necessity for reducing influenza-associated morbidity and mortality. Here we focus on the development of a universal influenza B virus vaccine based on the lipid nanoparticle-encapsulated nucleoside-modified mRNA (mRNA-LNP) platform. We evaluate vaccine candidates based on different target antigens that afford protection against challenge with ancestral and recent influenza B viruses from both antigenic lineages. A pentavalent vaccine combining all tested antigens protects mice from morbidity at a very low dose of 50 ng per antigen after a single vaccination. These findings support the further advancement of nucleoside-modified mRNA-LNPs expressing multiple conserved antigens as universal influenza virus vaccine candidates.

Phosphine-free ruthenium NCN-ligand complexes and their use in catalytic CO2 hydrogenation.

This work investigates the hydrogenation of carbon dioxide to formate catalysed by the phosphine-free Ru complexes Ru(OtBu)(κ3-NCN)(tBubpy) and RuH(OtBu)(κ2-NCN)(tBubpy) (OtBu = tert-butoxide, κ2-NCN = 1,3-di(2-methylpyridyl)-4,5-diphenyl-1H-imidazol-2-ylidene, where one pyridyl moiety is not coordinated to Ru, tBubpy = 4,4'-di-tert-butyl-2,2'-dipyridyl). A catalytic cycle is proposed for this reaction, supported by computational studies and the characterization of the hydride and the formate intermediates proposed to be involved. Modest catalytic turnovers are demonstrated at relatively low pressures and temperatures. The proposed rate determining step is heterolytic H2 splitting to regenerate the Ru-H complex, which has an estimated hydricity of approx. 27 kcal mol-1. The κ2-NCN ligand in the hydride complex undergoes a variety of dynamic processes as detected by EXSY spectroscopy including a pyridyl "roll-over" carbon-hydrogen - ruthenium hydride exchange, possibly occuring via a Perutz-Sabo-Etienne CAM mechanism.

Influence of particle size on the in vivo potency of lipid nanoparticle formulations of siRNA.

Lipid nanoparticles (LNP) can provide a clinically effective method for delivering small interfering RNA (siRNA) to silence pathological genes in hepatocytes. The gene silencing potency of these LNP-siRNA systems has been shown to depend on a variety of factors including association with serum factors such as ApoE and the pKa of component ionizable lipids. Here we investigate the influence of LNP size, an important parameter affecting tissue penetration of LNP systems, on the pharmacokinetics, biodistribution, and hepatic gene silencing potency of LNP-siRNA systems following intravenous administration. For LNP systems stabilized by a polyethylene glycol (PEG)-lipid that can dissociate from the LNP following injection, it is shown that small (diameter≤30nm) systems are considerably less potent than their larger counterparts. This is attributed in part to the ability of other lipid components, particularly the ionizable amino-lipid, to dissociate from the LNP following dissociation of the PEG-lipid. Small LNP stabilized by PEG-lipids with slow dissociation rates exhibited much reduced amino-lipid dissociation rates, however such systems are relatively impotent due to the continued presence of the PEG coating. These results demonstrate the delicate balance between the in vivo potency of LNP-siRNA systems and the residence times of component lipids in the LNP particle itself and suggest new directions to optimize the in vivo gene silencing potency of small LNP-siRNA systems.